RESEARCH ACCOMPLISHMENTS NOMID STUDIES: CLINICAL: 1. We analyzed 5 year outcome data in 20 patients and 3 year outcome data in 26 patients with NOMID who have been receiving escalating doses of anakinra. The initially observed good clinical response to anakinra persists, the drug is overall well tolerated. In most patients hearing and vision was preserved over 3 and 5 years, however patients who experienced hearing loss on anakinra had already significant hearing loss at baseline and more persistent enhancement on MRI. Low grade leptomeningeal inflammation was seen in up to 50% of patients at 3 years but dropped further at 5 years. Although no new bone lesions occurred with anakinra treatment, preexisting bony lesions continued to expand on treatment. These data suggest that treatment with anakinra not only controls disease symptoms and inflammatory blood markers but can also prevent the progression of organ damage. Studies to assess the prevention of any organ damage in very young children continue. 2. Disability on treatment with anakinra significantly improvement initially and we are currently characterizing persisted disabilities at 3 and 5 years. 3. An ongoing study in patients with NOMID using the long acting IL-1 inhibitor canakinumab with the goal of finding an optimal dose regimen and monitoring long term outcome is currently being analyzed. LABORATORY: 1. We studied chondrocyte primary cell lines obtained from a bone lesions (lesional)and articular cartilage (non-lesional) from one patient with NOMID during a surgical knee procedure. In collaboration with Dr. Stratakis laboratory (NICHD), we found that NOMID bone lesions are derived from the same osteoblast progenitor cells that form fibroblastoid tumors in mice and humans with defects that lead to increased cAMP-dependent protein kinase A (PKA) signaling. Lesional but not nonlesional cartilage cells in NOMID showed high PKA activity, and cAMP mediated PKA-specific caspase-1 activation. Caspase-1 activation via over-expression of the proto-oncogene Ets-1 is inflammation independent and led to increased prostaglandin E2 (PGE2) and cAMP levels and activation of Wnt signaling, similar to other states of inappropriate PKA activity. These findings suggest a molecular signaling pathway that can explain our clinical observations that NOMID bone lesions are non responsive to IL-1 blocking agents. 2. In collaboration with Dr Nishikomori's group in Kyoto an international case-control study was performed to detect somatic NLRP3 mosaicism in NOMID patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID patients and their healthy relatives. Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8%(mean SD 12.1 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18/26 vs. 0/19 P<0.0001). The detected somatic NLRP3 mutations had disease-causing functional effects. A lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurological symptoms. DIRA STUDIES: 1. In addition to the known founder mutations in DIRA, in Newfoundland, Holland, Puerto Rico and Lebanon, we identified a novel founder mutation in Brazil in collaboration with Drs. El-Shanti and Jesu. The 15 basepair in frame deletion leads to the encoding of a nonfunctional protein;patients have excellent responses to therapy and one patient had a loss of the odontoid of the second cervical vertebra secondary to a destructive inflammatory lesion. UNDIFFERENTIATED AUTOINFLAMMTORY DISEASES Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome is an autoinflammatory syndrome recently described in children. We investigated the clinical phenotype, genetic cause and the immune dysregulation in nine CANDLE patients in an international effort. We have clinically recently identified that CANDLE is caused by mutations in PSMB8, a gene that encodes an important protein degrading complex called i-proteasome. We identified two mutations in PSMB8, 6 patients were homozygous for the missense mutation (c.224C>T)and two patients were heterozygous for this mutation, one patient was homozygous for a novel nonsense mutation (c.405C>A) suggesting a protein truncation, one patient is mutation negative for PSMB8 mutations suggesting gentic heterogeneity. Mutations in PSMB8 were found concomitantly in adult patients clinically described as JMP (joint contractures, muscle atrophy and panniculitis induced lipodystrophy) syndrome in the US and by two Japanese groups. The detection of mutations in the same gene broadened the clinical genotype associated with this disease and showed that the early onset in our patients indicated a very severe subset presenting early in life. We extend the clinical and pathogenic description of this novel autoinflammatory syndrome. Functional studies showed that mutation-positive and mutation-negative patients expressed high IP-10 (Interferon gamma-induced protein 10) levels. Levels of MCP-1, IL-6, and IL-1Ra were moderately elevated. Microarray profiles and monocyte stat-1 activation suggested a unique interferon (IFN) signaling signature, unlike in other autoinflammatory disorders. We hypothesize that IFN may be a key inflammatory mediator of the inflammatory response in CANDLE and we plan to explore targeting this cytokine this as a therapeutic target. CONCLUSIONS AND SIGNIFICANCE 1. IL-1 blocking therapies have become the standard of treatment for patients with CAPS and DIRA and other autoinflammatory diseases with clinical similarities to the genetic IL-1 diseases. We continue to evaluate the long term safety and efficacy with these agents in our protocols. We are in the process of investigating whether long acting IL-1 blocking agents will also have a role in the treatment of DIRA. The understanding of the pathogenesis of these disorders provided key insights into the regulation of IL-1 as a key inflammatory cytokine in common disorders such as Type 2 diabetes, gout, obesity and coronary artery disease. 2. In collaboration with other groups we continue to search for mutations in patients with the clinical phenotype of NOMID who remain mutation negative. We search for the genetic causes in patients with autoinflammatory conditions not responsive to IL-1 blocking therapies to identify additional targets for therapeutic interventions. 4. Long-term treatment with IL-1 blocking therapy indicates that treatment of patients with NOMID is safe and effective over 5 years after initiation of treatment. Anakinra treatment improves disability and retards/stops progression of hearing loss and vision loss. 5. Treatment with long acting IL-1 inhibitors may allow for more convenient treatment options particularly in children in whom daily injections can be quite traumatic. A study evaluating the efficacy of the long acting IL-1 inhibitor canakinumab in patients with NOMID is being evaluated. 6. The evaluation of IL-1 blockade unresponsive patients led to the discovery of a novel autoinflammatory disease, CANDLE also called JMP, MMS and JASL that is caused by mutations in PSMB8 a gene encoding for a component of an immunoproteasome. The identification of a molecular defect in the protein processing machinery of a cell and our investigations regarding an inflammatory mechanism responsible for the disease phenotype will allow us to explore additional targets in the treatment of these patients. The prominence of an interferon signature may provide such a target for treatment but validation in the context of a treatment study is necessary.